Alu Element Genotypes

Alu item is a short run of DNA typically defined by the activity of Arthrobacter luteus (Alu) limited endonuclease. Alu elements seem to be the most common transposable elements, comprising more than a million copies spread across the human genome. Transposable elements are unusual DNA sequences that, within a single cell's genome, may transfer (or transpose) themselves to new positions. Alu elements are assumed to be parasite DNA or selfish since self-reproducing is their only known function. They are very likely to be playing an evolutionary role though and used as genetic markers. Therefore, if any two people have a similar inclusion of Alu components, we should conclude that this is probably because of common ancestry.

Alu elements come from the tiny cytoplasmic 7SL RNA, which is a portion of the particle of signal recognition. Alu elements are actively preserved in primate genomes and derived in a Supraprimates ancestor's genome.

The Alu insert in the region of PV92 is a way of marking a population's paternal and maternal history and reflecting unique evolutionary developments (Nasidze et al., 2001). While these repetitive sequences help map out specific features throughout the human genome, the origin and critical role of these sequences are still unknown. You will find Alu elements in the human genome in various locations; this, therefore, indicates primary evolutionary importance. The study of Alu elements in the PV92 area led researchers to trace Africa's modern human roots (Comas et al., 2001).

Alu insertions tend to explain genetic diversity across a population, as each Alu insertion occurring in that population is an extraordinary evolutionary occurrence, free of homoplasy (Comas et al., 2001), causing the Alu to replicate the same gene through descent from a shared ancestor (Batzer, 2002). Once inherited, alu polymorphisms stay the same, so it is easy to tell where some insertions come from. Alu elements are identifiers that aren't influenced by mutation rates, so with mutation and filtering omitted, Alu insertion analysis will represent the evolution of genetic drift and gene flow relationships in the past (Garcia-Obreon et al., 2007).

In 2008, research by Venkateswara Rao, Lakshmi, Demarchi, and Veerraju, found that there was a high degree of variance between Alu inserts among the five tribal populations examined. The significant disparity between people indicates that the insertions of Alu may help map out the relationship between different groups (Veerraju et al., 2008). Research on Iberian Basque populations also showed a significant gap between Caucasian, North Africa, and Europe in 2007 (Garcia-Obregon et al., 2007).

The PV92 area situated on chromosome 16, and dimorphic, as opposed to polymorphic, is the particular Alu insertion we will be examining. Alu sequences would typically have more than two phenotypes, but with this Alu sequence within the PV92 region, a person either has an allele present, or they do not have the allele present, and for each genotype, there is no apparent phenotype (Szczys, 2010). A polymerase chain reaction (PCR) is used to isolate the target sequence of PV92. In vitro, PCR is used to duplicate the specific piece of DNA in question. A combination of a DNA template, primers, buffer, magnesium ions, and free nucleotides, are required for a PCR to take place (Szczys, 2010). The answer is then followed by 40 cycles of three various steps, such as a denaturing step (94EšC), an annealing step (60EšC), and an extension step (72EšC). We're trying to use PCR to replicate our aimed sequence, so we've got more than enough DNA to do it.

The aim of the specific experiment we will be undergoing now is to use the Hardy-Weinberg Equilibrium method to decide if there has been any change in our data samples taken within the laboratory parts, or if we can acknowledge our false hypothesis that the population will be in HWE. We should conclude that our statistics show signs of evolution because our sample size is quite small and may represent a significant deviation. The three genotypes under review are homozygous positive (++) with the Alu insert present on both alleles, heterozygous (+-) with the Alu insert present on one allele, and not on the other, and eventually homozygous negative--) (with the Alu insert missing on both alleles.

Experiment

There was a collection of two hairs, trimmed to 2-3 cm comprising a coat of epithelial cells across their base and put in a 200μl InstaGene matrix + protease screw cap tube. For five minutes, the tubes were incubated at 56°C, vortexed, and then incubated for yet another five minutes. Then the tubes were put for five minutes in the boiling water bath (100°C). The tubes were vortexed and then for five minutes centrifuged at 6,000x g. It then moved 20μl of the supernatant to the base of a PCR tube and added 20μl of the master mix. The solution was combined with a pipette, and tubes were positioned for 40 amplification cycles in about 3 hours in the MyCycler thermal cycler.

The samples of the PCR were withdrawn from the thermal cycler, and 10μl PV92 XC loading dye and 10μl SYBR gold dye were added to one of the PCR tubes and softly combined. Then 20μl was extracted from each PCR tube and pumped inside the into individual wells within the previously prepared agarose gel For comparison purposes, molecular weight requirements, and DNA controls (+, +), (+,-),(-,-) have been mounted close to the samples. The gel was then tested for thirty-five minutes at 100 volts and then for another forty-five minutes at 200 volts. A photograph taken with UV light measured the distance traveled by the samples.

Outcome

The results were collected by calculating the number of students who displayed every one of the three genotypes. The DNA model used to represent DNA samples comprised of two homozygous samples (++)-) (and one heterozygous sample (+-). The bands of students who had two chromosomes with the insert were 941 base pairs in length, and the students who lacked the insert on both chromosomes had bands at 641 base pairs in length. Students with a heterozygous genotype had both a band in base pairs of 941 and a band in base pairs of 641. Among the 18 students in the study, one was homozygous positive, eight were heterozygous, and nine were homozygous negative.